The classical method
of quantification for wax esters achieved through hydrolysis of wax esters
after TLC separation and subsequent quantification of the derivatised fatty
acid and alcohol molecules by capillary column gas chromatography coupled with
flame ionisation detection (GC-FID) and GC-mass
spectrometry (GC-MS). High temperature GC capillary columns have
been used for the analysis and quantification of wax ester Molecular species
by GC-MS with some limitations such as difficulties in identification of
wax esters with polyunsaturated aliphatic side chains which exhibit very low
abundant molecular ions and low abundant diagnostic fragment ions (293, 294). Future
development is anticipated in the quantitative and molecular species analyses
of wax esters in various sample types using high efficiency capillary GC
columns stable at high temperature, and combination of columns with different
polarity in tandem, within GC-FID and GC-MS techniques.
pressure liquid chromatography (HPLC) coupled with atmospheric pressure
chemical ionization (APCI-MS), has been used for profiling the wax ester
species from different sources with limitations for quantification (295-298).
semi-quantitative method for profiling wax ester molecular species in seed oil
by a direct infusion nano-electrospray ionization tandem mass
spectrometry (nano-ESI-MS/MS), was developed (299).
The method has shown to be suitable to screen large numbers of samles of
different origins. By analyzing a large number of standard samples of wax
esters, the authors have shown that fragmentation patterns are highly dependent
on the double bond distribution between the fatty alcohol and the fatty acid
part of the wax ester. The mass spectral characteristics of the wax ester
standards will be useful in future studies for identification of molecular series
of these compounds (299).
The most widely used method for the
analysis of wax esters is GC, but prior to the GC analysis wax ester fraction
has to be isolated from the triglycerides by means of liquid chromatography
(LC) on silica gel, thin-TLC or by SPE (244). GC-MS has
proved an effective combination for chemical analysis, in order to carry out
simultaneous identification and quantification of the various components of
complex mixture. For some wax components, derivatization is required; as fatty
acids and fatty alcohols are not volatile, derivatization is necessary, but
fatty hydrocarbons are volatile and can be analyzed without derivatization (272).